In vitro stability and protein composition of thick filaments from insect flight muscles

Thick and thin filaments of synchronous and asynchronous insect flight muscles were separated by density gradient centrifugation. A good release of myofilaments from myofibrils was obtained by sonication of myofibrils in relaxing solution with pH 6.1 (locust), pH 6.4 (honeybee) and pH 6.6 (fleshfly), respectively. Thick filaments but not thin filaments were dissolved, if sucrose gradient centrifugation was used to separate the filaments. Thus, sucrose gradients are the medium of choice if actin filaments are to be purified. Glycerol-containing gradients selectively dissolved myosin filaments from fleshfly muscles. The stability of the myosin filaments of all muscles was sufficient in gradients with 10–30% formamide.     

Immunocytochemical demonstration of contractile cells in the human ovarian follicle

Summary Actin-and myosin-like immunoreactivity is found in cells located in the theca externa of the follicle wall of the human ovary, and corresponding to previously observed myoid cells. The immunocytochemical observation provides direct structural evidence that non-vascular contractile cells are also present in the follicle wall in humans. As expected, perifollicular blood vessels showed a positive immunoreaction for actin and myosin in their smooth muscle walls.This work was supported by the Swedish Medical Research Council, grant No. 14X-732/5680.     

Molecular motors: not quite like clockwork

Models commonly used to explain the mechanism of myosin motors typically include a power stroke that is attributed to a conformational change in the motor domain and amplified by a long lever arm that connects the motor domain to the cargo. Similar models have proved less enlightening in the case of microtubule motors, for which it may be more helpful to consider models involving thermally driven mechanisms. Received 9 November 2007; received after revision 7 December 2007; accepted 11 December 2007     

Drosophila unconventional myosin VI is involved in intra- and intercellular transport during oogenesis

During mid-oogenesis of Drosophila, cyto plasmic particles are transported within the nurse cells and through ring canals (cytoplasmic bridges) into the oocyte by means of a microfilament-dependent mecha nism. Video-intensified fluorescence timelapse mi croscopy, in combination with microinjections of antibodies directed against Drosophila 95F myosin, have revealed that this unconventional myosin of class VI is involved in the transport processes. The results indicate that certain cytoplasmic particles in the nurse cells move along microfilaments due to their direct association with myosin VI motors. Additional myosin- VI molecules located at the rim of the ring canals seem to be involved in particle transport into the oocyte. Microinjected mitochondria-specific dyes have revealed that some of these particles are mitochondria. Received 3 April 1997; received after revision 5 May 1997; accepted 27 May 1997     

TheDictyostelium cytoskeleton

New avenues of cytoskeleton research inDictyostelium discoideum have opened up with the cloning of the - and -tubulin genes and the characterization of kinesins and cytoplasmic dynein. Much research, however, continues to focus on the actin cytoskeleton and its dynamics during chemotaxis, morphogenesis, and other motile processes. New actin-associated proteins are being identified and characterized by biochemical means and through isolation of mutants lacking individual components. This work is shedding light on the roles of specific actin assemblies in various biological processes.     

Minoxidil-induced cardiac hypertrophy in guinea pigs

To investigate whether during cardiac hypertrophy changes occur in contractile protein composition and in mechanical and energetic properties of the myocardium, contractile protein composition, isometric force and adenosine triphosphate (ATP) consumption were studied in control and hypertrophied guinea-pig hearts. Cardiac hypertrophy was induced by adding minoxidil (120 or 200 mg/l) to the drinking water. Protein analysis was performed by one-dimensional gel electrophoresis. The myosin heavy-chain (MHC) composition was determined in an enzyme-linked immunosorbent assay (ELISA). ATP consumption and force development were simultaneously measured during isometric contraction in chemically skinned trabeculae. Histochemical analysis of cross-sectional area of cardiomyocytes and interstitial space was performed on the left ventricular tissue of 200 mg/l minoxidil-treated and control guinea pigs. Minoxidil treatment (120 and 200 mg/l) significantly increased left ventricular dry weight normalized for body weight by 19 ± 4 and 24 ± 4%, respectively. No significant differences were found in the cellular cross-sectional area, while interstitial space was slightly decreased in minoxidil-treated hearts. In left ventricular trabeculae of 200 mg/l minoxidil-treated guinea pigs, ATPase activity was slightly less than in those of control guinea pigs, whereas force did not differ significantly. Calcium sensitivity of force and ATPase activity were not affected by minoxidil treatment. Gel electrophoresis revealed no difference in contractile protein composition, but a tendency towards a lower amount of α-MHC in the minoxidil-treated hearts was found in ELISA. Received 1 February 1999; accepted 15 March 1999     

The diversity of molecular motors: an overview

Rapid progress has recently been made in the identification and characterization of a large number of kinesin and myosin motor proteins. Recent work has uncovered roles for these motors in processes such as vesicle trafficking, cytoskeletal organization, and chromosome movements, to name a few. A series of reviews describing some of the significant advances in our understanding of the structure and function of myosins and kinesins is presented.     

Myosins from plants

Molecular cloning and sequence analysis of myosin genes from Arabidopsis thaliana and electron microscopic observation of a myosin from characean alga have revealed that overall structure of plant unconventional myosins is similar to that of the class V myosins. These plant unconventional myosins have two heads, a coiled-coil tail of varied length and a globular tail piece at the end. The tail piece is probably a site for membrane interaction. Characean myosin is of special interest because it can translocate actin filaments at a velocity several times faster than muscle myosin, which must have evolved to support the quick movement of animals in the struggle for their lives.     

The Ras family of GTPases in cancer cell invasion

The ability of tumoral cells to invade surrounding tissues is a prerequisite for metastasis. This is the most life-threatening event of tumor progression, and so research is intensely focused on elucidating the mechanisms responsible for invasion and metastasis. The Ras superfamily of GTPases comprises several subfamilies of small GTP-binding proteins whose functions include the control of proliferation, differentiation, and apoptosis, as well as cytoskeleton organization. The development of metastasis is a multistep process that requires coordinated activation of proliferation, motility, changes in normal cell-to-cell and cell-to-substrate contacts, degradation of extracellular matrix, inhibition of apoptosis, and adaptation to an inappropriate tissue environment. Several members of the Ras superfamily of proteins have been implicated in these processes. The present review summarizes the current knowledge in this field.     

The role of myosin phosphorylation in the contraction-relaxation cycle of smooth muscle

Summary Considerable evidence from a variety of experimental procedures indicates that the phosphorylation of myosin is involved in the regulation of contractile activity in smooth muscle. Phosphorylation of the 20,000-dalton myosin light chains is required to initiate crossbridge cycling and this is consistent with the observation that the actin-activated Mg²⁺-ATPase activity of myosin is phosphorylation-dependent. In the simplest interpretation of this process it may be proposed that phosphorylation acts as an on-off switch. Clearly this cannot explain the observed complexity of smooth muscle contractile behavior and such may imply either that additional mechanisms are involved or that the role of myosin phosphorylation is not fully appreciated. Recently it has been shown that monomeric smooth muscle myosin can exist in a folded and an extended conformation and that each form is characterized by distinct enzymatic properties. Under appropriate solvent conditions phosphorylation of myosin favors the extended conformation. It is tentatively suggest that this, or an analogous, transition might be involved in the regulation of the smooth muscle contractile apparatus, and this possibility is discussed.The authors are supported by grants HL 23615 and HL 20984 from the National Institutes of Health.     

Signalling roles of mammalian phospholipase D1 and D2

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidate (PA) and choline. PLD activity in mammalian cells is low and is transiently stimulated upon activation by G-protein-coupled and receptor tyrosine kinase cell surface receptors. Two mammalian PLD enzymes (PLD1 and PLD2) have been cloned and their intracellular regulators identified as ARF and Rho proteins, protein kinase Cα as well as the lipid, phosphatidylinositol [4, 5] bisphosphate (PIP₂). I discuss the regulation of these enzymes by cell surface receptors, their cellular localisation and the potential function of PA as a second messenger. Evidence is presented for a role of PA in regulating the lipid kinase activity of PIP 5-kinase, an enzyme that synthesises PIP₂. A signalling role of phospholipase D via PA and indirectly via PIP₂ in regulating membrane traffic and actin dynamics is indicated by the available data. Received 25 April 2001; received after revision 15 June 2001; accepted 15 June 2001     

Biological effects of lithium: Experimental analysis in plant cytokinesis

Summary The biological effects of lithium ions have been studied, using plant cytokinesis in onion root meristems as the experimental model. Lithium induces binucleate cells by inhibiting cell plate formation. Moreover, lithium and caffeine have additive effects on the induction of binucleate cells. Na⁺, K⁺, Ca⁺⁺ and Mg⁺⁺ antagonize lithium-induced inhibition of cytokinesis.     

Myosin regulation in the migration of tumor cells and leukocytes within a three-dimensional collagen matrix

The migration of cells is a complex regulatory process which results in the generation of motor forces through the reorganization of the cytoskeleton. Here we present a comparative study of the expression and involvement of myosin in the regulation of the physiological migration of leukocytes and the pathological migration of tumor cells. We show that the involvement of myosin in the migration is distinct in these two cell types. In leukocytes, the activity of non-muscle myosin II is essential for both the spontaneous (matrix-induced) migration and the migration induced by ligands to G protein-coupled receptors, i.e. chemokines and neurotransmitters. In contrast, spontaneous tumor cell migration is largely independent of non-muscle myosin II activity, whereas the norepinephrine-induced migration is completely inhibited by either direct inhibition of non-muscle myosin II or of the kinases phosphorylating the myosin light chain, namely ROCK or the calcium/calmodulin-dependent myosin light-chain kinase.Received 31 August 2004; accepted 26 October 2004     

Regulation of B and T cell development by anterior pituitary hormones

Hormones produced by the anterior pituitary gland have been implicated in the regulation of primary lymphocyte development. In order to identify endocrine factors involved in that process, several strains of mice with genetic defects resulting in a selective impairment in the production of one or more anterior pituitary-derived hormones have been analysed. This study has resulted in the classification of endocrine hormones into the following four categories (i) hormones such as prolactin with no apparent effects on primary lymphopoiesis; (ii) anabolic hormones such as growth hormone and insulin-like growth factor-I whose stimulatory effects on primary lymphopoiesis are non-lineage-specific and related to their actions as systemic mediators of growth and/or differentiation; (iii) hormones such as thyroid hormones that have an obligate role in primary B lymphopoiesis; and (iv) hormones such as oestrogens that act as negative regulators of lymphopoiesis.     

Four cross-bridge strands and high paramyosin content in the myosin filaments of honey bee flight muscles

Summary Measurements of the mass ratio of myosin to paramyosin of myofibrils of honey bee flight muscles on sodium dodecyl sulphate-polyacrylamide gels yielded a paramyosin content of 24% of the myosin filament mass. Based on the myosin to actin mass ratio of 2.3, and 3 actin filaments per myosin filament and per half sarcomere, it could be calculated that there were 3.8 myosin molecules repeating regularly at intervals of 14.4 nm along the myosin filament. In spite of the high paramyosin content the diameter of the myosin filaments is 19–20 nm, as in other insect flight muscles.     

Intramuscular comparison of myosin isozymes and light chains in rat extensor digitorum longus muscle

Summary Complete muscle cross sections were obtained from the proximal and distal third regions of ten rat extensor digitorum longus muscles. Electrophoretic methods were then used to quantify the various myosin isozymes and light chains in each muscle specimen. The results demonstrated that the relative distribution of the various myosin isozyme and light chain variables do not vary significantly between the two sampling regions.     

Regulation of G1 phase progression by growth factors and the extracellular matrix

Cell cycle progression is regulated by both intracellular and extracellular control mechanisms. Intracellular controls ensure that cell cycle progression is stopped in response to irregularities such as DNA damage or faulty spindle assembly, whereas extracellular factors may determine cell fate such as differentiation, proliferation or programmed cell death (apoptosis). When extracellular factors bind to receptors at the outside of the cell, signal transduction cascades are activated inside the cell that eventually lead to cellular responses. We have shown previously that MAP kinase (MAPK), one of the proteins involved in several signal transduction processes, is phosphorylated early after mitosis and translocates to the nucleus around the restriction point. The activation of MAPK is independent of cell attachment, but does require the presence of growth factors. Moreover, it appears that in Chinese hamster ovary cells, a transformed cell line, growth factors must be present early in the G1 phase for a nuclear translocation of MAPK and subsequent DNA replication to occur. When growth factors are withdrawn from the medium immediately after mitosis, MAPK is not phosphorylated, cell cycle progression is stopped and cells appear to enter a quiescent state, which may lead to apoptosis. Furthermore, in addition to this growth-factor-regulated decision point in early G1 phase, another growth-factor-sensitive period can be distinguished at the end of the G1 phase. This period is suggested to correlate with the classical restriction point (R) and may be related to cell differentiation.